An efficient human stem cells derived cardiotoxicity testing platform for testing oncotherapeutic analogues of quercetin and cinnamic acid

Oncotherapeutics research is progressing at a rapid pace, however, not many drugs complete the successful clinical trial because of severe off-target toxicity to cardiomyocytes which ultimately leads to cardiac dysfunction. It is thus important to emphasize the need for early testing for possible cardiotoxicity of emerging oncotherapeutics. In this study, we assessed a novel stem cell-derived cardiac model for testing for cardiotoxicity of novel oncotherapeutics. We evaluated the cardiotoxic effect of synthesized derivatives of oncotherapeutics, quercetin (QMJ-2, -5, and -6) and cinnamic acid (NMJ-1, -2, and -3) using human Wharton's jelly mesenchymal stem cells-derived cardiomyocytes (WJCM) against known cardiotoxic oncologic drugs, doxorubicin, 5-fluorouracil, cisplatin. QMJ-6, NMJ-2, and NMJ-3 were not cardiotoxic and had minimum cardiac side effects. They did not show any effect on cardiomyocyte viability, caused low LDH release, and intracellular ROS production kept the calcium flux minimal and protected the active mitochondrial status in cardiomyocytes. They persevered cardiac-specific gene expression as well. However, compounds QMJ-2, QMJ-5, and NMJ-1 were cardiotoxic and the concentration needs to be reduced to prevent toxic effects on cardiomyocytes. Significantly, we were able to demonstrate that WJCM is an efficient cardiac testing model to analyze the cardiotoxicity of drugs in a human context.

Cancer is a multifactorial disease 1 and due to tremendous advancement in treatment modalities such as surgery, chemotherapy, and radiation therapy, mortality has declined followed by improvement in survivorship 2 . However, cardiovascular diseases (CVDs) have emerged as major side effects among cancer survivors that may lead to long-term morbidity and premature deaths 3 . This is related to the outcome of cardiotoxicity resulting directly from the cancer treatment and that could have compromised the cardiac function and structure 4 .
Over time, a large number of the anti-cancer drug has been withdrawn from the market and the reason was an unintended effect on cardiac health 3 . The anthracycline class of chemotherapeutic drugs which consists of doxorubicin, 5-fluorouracil, paclitaxel, cisplatin, idarubicin, epirubicin, and mitoxantrone are known for exerting an adverse cardiotoxic effect on cardiomyocytes 5 . In 2016, the European Society of Cardiology (ESC) suggested a prior check to identify the risk of developing cardiovascular toxicity by any cancer treatments and therapy. Treatment modifications can thus be practiced that may allow decreasing the risk for further cardiotoxicity before administration to the patients 6 .
In search of a new therapeutic agent, chemically synthesized compounds whose structures mostly are inspired by natural compounds have been explored enormously against various disease models 7 . Bringing any newly synthesized anti-cancer molecule even after extensive preclinical testing, the entity faces several challenges to get translated for clinical utility because of their potential to cause cardiotoxicity 8  Treatments. WJMSC-derived cardiomyocytes (WJCM) and H9C2 were treated with NMJ and QMJ analogues for 24 h. The concentration of these compounds was based on their previously reported IC 50 values which were obtained using Vero, a non-cancerous cell line i.e., QMJ-2 (140 µM), QMJ-5 (55.6 µM), QMJ-6 (153 µM), NMJ-1 (9.7 µM), NMJ-2 (8.2 µM), NMJ-3 (15.1 µM) 43,44 . To illustrate the toxicity specific to cardiac cells and not a general cytotoxicity, WJMSCs were treated with positive cardiotoxic drugs. The positive cardiotoxic drugs used were doxorubicin (Dox) 46 , 5-fluorouracil (5FU) 47 , and cisplatin (Cis) 48 .
Cell viability assays. WJMSCs were treated with Dox (1 µM), 5FU (1 µM), and Cis (5 µM Lactate dehydrogenase (LDH) assay. LDH activity was observed in WJMSCs treated with positive cardiotoxic drugs as well as in the H9C2 cells and WJCM after they were exposed to QMJ and NMJ analogues using Pierce LDH cytotoxicity assay kit (Thermo Scientific, MA, USA Gene expression analysis. Total ribonucleic acid (RNA) was isolated using RNAiso Plus (TaKaRa, Shiga, Japan) followed by cDNA conversion using PrimeScript™ 1st strand cDNA synthesis kit (TaKaRa, Shiga, Japan). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed by using TB Green™ Premix Ex Taq™ II (Tli RNase H Plus; TaKaRa, Shiga, Japan) for cardiac markers in a QuantStudio™ 5 real-time PCR Systems (Applied Biosystems, USA). All the primers were purchased from Sigma Aldrich, Bangalore, India ( Table 1). The relative gene expression was calculated using 2 -∆∆CT and GAPDH, a housekeeping gene was used as endogenous control 52,53 . Statistical analysis. Data were analyzed in GraphPad Prism v8 (GraphPad Software, Inc., CA, USA) and are expressed as mean ± SEM. Student's t-test or one-way ANOVA followed by Dunnett's post-test was performed. Data were considered statistically significant when p < 0.05.

Status of ROS generation and NO release upon treatment with quercetin and cinnamic acid analogues.
Excessive reactive oxygen species (ROS) is one of the main sources of cell damage which leads to cardiotoxicity. We thus measured ROS activity in H9C2 cells and WJCM after exposure to the drug analogues. Similar to previous results, both QMJ-6 and NMJ-3 were not toxic to the cells, and along with NMJ-2 in WJCM, there was lowered ROS activity whereas an increase in ROS activity was detected after treatment with the rest of the analogues (Fig. 3a,d). Furthermore, we also estimated the levels of nitric oxide (NO), a known vasodilator 54 by using a modified Griess reagent. QMJ-6 and NMJ-2 in H9C2 cells (Fig. 3b) and NMJ-2 in WJCM elevated NO levels while NMJ-3 decreased NO level by 12% WJCM (Fig. 3e). Rest of the analogues did not show much effect.
Cardiotoxic effect of quercetin and cinnamic acid analogues on calcium accumulation. Initiation of myocardial injury and impairment of contractile function is thought to be caused due to accumulation of intracellular Ca 2+ . Here, we observed a decrease in Ca 2+ accumulation after exposure of cells to QMJ-6, NMJ-2, and NMJ-3 in both H9C2 and WJCM cells (Fig. 3c,f). The other analogues studied displayed cardiotoxicity similar to Dox (in WJCM) or higher calcium accumulation (in H9C2).
Modulation of mitochondrial membrane potential after treatment with quercetin and cinnamic acid analogues. Depolarization of mitochondrial membrane and associated ROS production leads to induction of apoptosis in cells. We observed increased mitochondrial membrane potential using TMRE in H9C2 cells treated with QMJ-6, NMJ-2, and NMJ-3 ( Fig. 4a,b) and WJCM treated with QMJ-6 and NMJ-3 (Fig. 4c,d) detected quantitatively and by fluorescent microscopy.

Expression of cardiac markers after treatment with quercetin and cinnamic acid analogues in WJCM.
To further investigate the cardiotoxic effect of these compounds in human-derived cardiomyocytes, we exposed WJCM with quercetin and cinnamic acid analogues followed by qRT-PCR analysis for cardiac genes.

Discussion
For many years, clinical data have substantially demonstrated that cancer drug-induced cardiotoxicity among cancer survivors leads to the loss of cardiomyocytes which ultimately compromises heart function 55 . There is a need for testing the hidden cardiotoxicity of any drug molecule before administering it to patients which will also increase the success rate of the drug marketing process. We thus need a suitable human-based model to test the cardiotoxicity. In this present study, we have established a cardiomyocytes model derived from human WJMSCs and assessed the utility of cardiomyocytes in monitoring the cardiotoxic events of novel anticancer drug molecules at the cellular and functional level. The advantage of using WJMSCs is that it is isolated using a non-invasive method and from a surgically discarded tissue. We have optimized an easy differentiation procedure to generate cardiomyocytes. These differentiated cells can be further cultured and maintained in a hassle-free manner. Our lab has previously reported the isolation of WJMSCs and their differentiation into functional cardiomyocytes 23,53 . Along with primary cardiomyocytes, based on the characteristics features, we considered H9C2 cells to understand and compare the impact of cardiotoxicity in an immortalized cell line with primary differentiated cardiomyocytes. For instance, the presence of cytoskeletal protein, beta-tubulin II, and their interactions with mitochondria is very crucial for energy metabolism in the heart. Kuznetsov et al. 32 attempted to study the difference in bioenergetics, and metabolic and functional characteristics between HL-1 (adult immortalized atrial cells) and H9C2 cells. It was found that beta-tubulin II was absent in HL-1 but present in H9C2 cells which suggest that H9C2 cells are bioenergetically/biochemically similar to primary cardiomyocytes. The heart requires an extensive amount of energy to carry out the function of blood pumping and 80% of the energy is supplied by mitochondria. The study revealed that the H9C2 cell has a content of mitochondrial mass, increased respiration rate, and enhanced ATP level in comparison to HL-1 cells 32 .
We investigated the effects of 3-hydroxyflavone (QMJ-2, QMJ-5, and QMJ-6) and cinnamyl sulfonamidehydroxamate (NMJ-1, NMJ-2, NMJ-3) in WJCM and H9C2 cells using the concentrations reported for noncancerous Vero cells. The prerequisite for any synthesized drug molecules is that they should be nontoxic to cardiac cells. We could show by MTT, CCK-8, and LDH assay that QMJ-6, NMJ-2, and NMJ-3 had no cytotoxic effect against cardiomyocytes whereas QMJ-2, QMJ-5, and NMJ-1 had induced toxicity suggesting that QMJ-2, QMJ-5, and NMJ-1 that these drugs could have an off-target cardiotoxic effect in vivo. www.nature.com/scientificreports/ The cancer drug-induced toxicity in patients is still an unavoidable clinical hurdle. The root of the problem is at the cellular level when chemotherapeutic drugs fail to differentiate between normal cells and tumor cells and indiscriminately target the normal cells causing undesired cytotoxicity. Therefore, a representative in vitro toxicity model is needed to understand the difference between target and non-target cells and then predict the possible toxicity 56 .
In our study, we checked the ability of our model to sense the difference between target and non-target cells, we performed cytotoxicity assays in undifferentiated Wharton's jelly mesenchymal stem cells considering them as normal cells to test the effect of chemotherapeutic drugs. We treated the WJMSCs with Dox, 5FU, and Cis at the same concentration used for cardiomyocytes. Interestingly, we found that these drugs do not exert any toxicity to WJMSCs and it shows that the model predicting the toxicity is specific to cardiac cells. Furthermore, studying primary cardiomyocytes provides many inherent benefits over whole-heart models whereas H9C2 cells have been the workhorse of in vitro cardiotoxic studies, both offering thorough control of experimental conditions including functionality, bioenergetics, metabolism, and genetic manipulations.
ROS at low amounts acts as "redox messengers" in the proliferation, survival, and differentiation of the cell. Meanwhile, disruption of mitochondria produces high ROS that ultimately becomes a reason for apoptosis 57 . During cardiotoxicity, elevated ROS alters physiological signaling responses which cause cellular hypertrophy, ventricular remodeling, sarcomeric instability, and calcium homeostasis, all these phenomena eventually induces cardiomyocytes death 58 . ROS generation damages the DNA and reduces the DNA binding activity of GATA binding protein 4 (GATA4), a critical cardiomyocytes transcriptional factor 59 . Our study showed that the ROS level remained unaffected in QMJ-6, NMJ-2, and NMJ-3 treated cardiomyocytes, however, QMJ-2, QMJ-5, and NMJ-1 treatment increased ROS levels that ultimately reduced cell viability. Dox generates ROS as a prime mechanism to induce toxicity against cardiomyocytes 60 . In the present study, increased ROS level in dox treatment is in agreement with the established mechanism.
Nitric oxide is a vasodilator, modulator of blood pressure, vascular tone, and hemodynamics 61 and NO addition promoted differentiation of embryonic stem cells into myocardial cells 62,63 . Production of NO gets altered by the binding of Dox leading to superoxide formation 10 . In our studies, we could demonstrate that Dox addition had reduced NO production, however, the synthesized compounds have not caused any changes in NO levels which indicates that NO physiology remains unaffected.
Cardiotoxicity induces dysregulation of intracellular calcium levels within cardiac cells that imply a cardiac remodeling. The toxicity suppresses mitochondrial respiration causing a low ATP state, which in turn slows the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) which causes an increase in diastolic potential. This change in intracellular Ca 2+ homeostasis is due to Ca 2+ unloading from the sarcoplasmic reticulum 64 which might lower the heart systolic potential and increase the chance of sudden cardiac arrest [65][66][67] . In our study, we observed that the intracellular calcium level remained low in QMJ-6, NMJ-2, and NMJ-3 treated cardiomyocytes, whereas QMJ-2, www.nature.com/scientificreports/ QMJ-5, and NMJ-1 exposure has increased the level indicating dysregulation of calcium homeostasis. Dox promotes the calcium accumulation in the mitochondria via the opening of mitochondrial permeability transition pores which alters the calcium balance 68 . Our result confirms the alternation in calcium due to Dox treatment. Mitochondrial-mediated cell death has emerged as a major mechanism for the death of cardiomyocytes. Cardiotoxicity disrupts mitochondria and their membrane potential which generates oxidative stress causing excessive ROS production in cardiomyocytes 58 . Upon treatment of cardiomyocytes with QMJ-6, NMJ-2, and NMJ-3, we observed that active mitochondria status remains unaffected whereas, in QMJ-2, QMJ-5, and NMJ-1 treatment cardiomyocytes displayed a loss of mitochondria. However, Dox treatment affected cardiomyocyte mitochondrial activity in concordance with the previous report 60 .
Cardiotoxicity drastically impacts the expression of cardiac genes such as NKX2.5, GATA4, cardiac troponins I and T, and myosin heavy chain alpha which are the functional markers of cardiomyocytes and can be used to assess cardiomyocyte's health and predict cardiac injury 69,70 . Troponins are the gold standard biomarkers that appear in blood with an increase in level within 2-3 h after myocardial damage due to cardiotoxicity 71 . A clinical study found various troponin I release pattern in breast cancer patients before and after chemotherapy where the major number of patients after chemotherapy had increased troponin I level suggesting that they could have a higher incidence of adverse heart effects and a greater left ventricular ejection fraction reduction as compared with patients who had little increase in troponin I level 72 . In the current study, using WJCM, we could see that similar other parameters assessed for cardiotoxicity, QMJ-2, QMJ-5, and NMJ-1 had a drastic reduction in the cardiac marker gene expression, similar to the cardiotoxic effect of Dox, 5FU, and Cis whereas QMJ-6, NMJ-2, and NMJ-3 had no significant impact on cardiac-specific genes. Thus, it is evident that across the parameters analyzed, quercetin and cinnamic acid derivatives QMJ-2, QMJ-5, and NMJ-1 are cardiotoxic, similar to the known cardiotoxic oncotherapeutics Dox, 5FU, and Cis whereas derivatives QMJ-6, NMJ-2, and NMJ-3 do not display significant cardiotoxicity. Furthermore, the compounds QMJ-2, QMJ-5, and NMJ-1 at a concentration of 68 μM, 27.4 μM, and 5.07 μM respectively found effective against cancer could be also actually toxic to cardiomyocytes. We considered the concentrations based on previously reported studies. In the previous studies, the in vitro cytotoxicity assay in Vero cells revealed that the treatment of derivatives QMJ-2, QMJ-5, QMJ-6, NMJ-1, NMJ-2, and NMJ-3 at concentrations of 140 µM, 55.6 µM, 153 µM, 9.7 µM, 8.2 µM, and 15.1 µM respectively reduces the cell viability by 50%. Vero cells are one of the most commonly used mammalian cell lines to study drug efficacy. Given that, we used the same concentrations to test the cardiotoxic effect in our model. Our observation suggests that the cardiotoxicity was specific to the derivative and not an outcome of concentrations. For example, NMJ-1 which was of quite low concentration i.e., 9.7 µM showed cardiotoxicity whereas QMJ-6 www.nature.com/scientificreports/ at a concentration of 153 µM was found non-cardiotoxic. The data obtained thus justified the purpose of the model which is to detect the cardiotoxicity exerted by any compound and the results are highly comparable with positive controls. To reduce the cardiotoxicity of these analogues, optimum concentration will have to be fixed which is effective against cancers and is not toxic to cardiomyocytes. The analogues QMJ-2, QMJ-5, and QMJ-6 are the result of substitutions in the 3-hydroxyflavone, a backbone of all flavonols whereas NMJ-1, NMJ-2, and NMJ-3 have been obtained after the modification based on bio-isosterism in the cinnamic acid moiety. Specifically, QMJ-2, QMJ-5, and QMJ-6 are 3-hydroxyflavone analogue with a methyl, di-methyl, and methyl and a methoxy substitution on the phenyl ring respectively 44 . NMJ-1, NMJ-2, and NMJ-3 are cinnamyl sulfonamide hydroxamate derivative with thiophene methyl amine, thiophene ethyl amine, and furfurylamine substitution respectively 43,45 . With various fundamental medicinal chemistry principles such as bio-isosterism, a wide range of chemical compounds are being synthesized with the purpose to augment absorption, distribution, metabolism, excretion, and toxicity (ADMET), improving efficacy, and enhancing chemical accessibility 73 . Fluorouracil is itself a chemical modification 74 . Furthermore, to overcome the issue of drug resistance and cardiotoxicity, Dox 75 has undergone several structural modifications, and new drug entities such as epirubicin, pirarubicin, valrubicin, and idarubicin have been synthesized 73 . The chemical modification brings significant therapeutic consequences and makes the lead compound highly relevant biologically 76 . In the Dox structure, the lipophilic part demonstrated acidic and hydrophilic amine sugar part showed basic nature which makes them soluble in both lipophilic and hydrophilic types of solvents so they bind to the plasma protein and cell membrane. Upon entering into the cells, Dox intercalates into nucleic acids and inhibits topoisomerase II which explains the mode of action. During the cell-killing action, excessive calcium release and dysregulation of mitochondrial function leads to the undesired outcomes 77,78 . Similarly, the analogues used in this study went through different chemical substitution with methyl and amine groups which may impact the molecular lipophilicity and cellular uptake and these could be possible reasons behind the cardiotoxicity as an off-target effect. However, it would be very interesting to study further the association of structural modification and its off-target effect.
A limitation of the study is that we have only assessed three chemotherapeutic drugs (Doxorubicin, 5-fluorouracil, and cisplatin). There are other classes of chemotherapeutic drugs which exerts cardiotoxicity, as well as various potential natural compounds which may have hidden cardiotoxic effect and so analysing their cardiotoxicity in our model, might have given new prospect. To establish the cardiotoxic effect, we have included an established cell line, H9C2 cells for comparison. Likewise, iPSCs-derived cardiomyocytes have been widely used to study cardiotoxicity. An inclusion of iPSCs would have made the comparison more exhaustive, however, iPSCs hold a set of challenges for disease modelling including cardiovascular diseases 79,80 . It has been observed that experimental and genetic variability in iPSCs can be overcome up to a certain limit but iPSCs culturing and maintenance is expensive and remains out of budget for many research labs across the world. Regarding animal studies, because of shortcomings like high cost, interspecies variability, and ethical concerns, the demand for an alternative to animal use is rising 14 .
In our study, we tested synthetic compounds which have already shown anti-cancer effects. Our model further indicates that the concentrations of few compounds which exerted anticancer effect could possibly show cardiotoxic effect too. We included experiments to study cardiotoxicity which result in cardiomyocyte death which is mainly mitochondria-mediated apoptosis. We believe that our model and experiments can be used to study hidden cardiotoxicity of other synthesized compounds, natural compounds, drugs molecules, etc.

Conclusion
In summary, by studying the oncotherapeutic molecules in human stem cells-derived cardiomyocytes we could effectively ascertain the varying cardiotoxic response of these molecules. From a perspective of human origin, human stem cells-derived cardiomyocytes could be a perfect preclinical cell-based model to study cardiotoxic events and reduce the dependence on animal usage in drug safety research.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. www.nature.com/scientificreports/